Process for preparing conjugated linoleic acid

ABSTRACT

The invention relates to a process for preparing conjugated linoleic acid. In particular, the invention discloses a process for preparing conjugated linoleic acid, particularly the cis-9,trans-11 isomer thereof from grain by means of beneficial bacteria. The invention also relates to the products prepared by the process.

FIELD OF THE INVENTION

The invention relates to a process for preparing conjugated linoleicacid. In particular, the invention discloses a process for preparingconjugated linoleic acid and in particular the cis-9,trans-11 isomerthereof from grain by means of beneficial bacteria.

BACKGROUND OF THE INVENTION

CLA is a generic term for different isomers of conjugated linoleic acid,of which only two isomers (cis-9,trans-11 isomer, i.e. bovine acid, andtrans-10,cis-12 isomer) have been found to be biologically active.Synthetic CLA products are commercially available but these usuallyinclude all different isomers of CLA and only 40% of c9,t11 isomer.Also, animal products, such as meat and milk, can be used as the CLAsource. An advantage of these is that most of the CLA they contain isc9,t11 isomer, e.g. milk CLA contains 80% of c9,t11-18:2 isomer.

Several studies have shown that animal fats include a fatty acid whichprevents, cancer in test animals, affects growth factors and mayregulate the amount of body fat tissue. When researching hamburgersteaks, Michael Pariza found that they include a fatty acid which wasanalyzed as conjugated linoleic acid (CLA). In studies carried out ontest animals, it was found that the occurrences of breast cancer,gastric cancer and cancer of the large intestine had decreased in thegroup fed with food containing CLA compared to the control group(Pariza, M. W., Loretz, L. J., Storkson, J. M. and Holland, N. C. 1983.Mutagens and modulator of mutagenesis in fried ground beef. Cancer Res.(Suppl.) 43: 2444-2446, and Pariza, M. W, and Hargraves, W. A. 1985. Abeef-derived mutagenesis modulator inhibits initiation of mouseepidermal tumors by 7,12-dimethylbenzanthracene. Carcinogenesis6:591-593). Also, CLA has been able to inhibit development of cancercells in tissue cultures of human cells. The mode of action is stillunknown, but CLA has been found to have influence on the differentdevelopment stages of cancer, several growth factors and possibly alsoon metabolism of carcinogenic substances in the liver. It has also beensuggested that CLA would function as an antioxidant (Ip, C., Chin, S.F., Scimeca, J. A., and Pariza, M. W. 1991. Mammary cancer prevention byconjugated dienoic derivatives of linoleic acid. Cancer Res.51:6118-6124), in which case the compound would protect cell membranesfrom the adverse effects of free radials. In addition, the decreasingeffect of the compound on the cholesterol level has been studied, and ithas been found that the compound does not decrease the amount of goodHDL as the cholesterol decreasing pharmaceuticals do (Lee, K. N.,Kritchevsky, D., and Pariza, M. W. 1994. Conjugated linoleic acid andatherosclerosis in rabbits. Atherosclerosis 108:19-25). CLA may alsohelp weight-watchers since the compound has been found to degrade fattissue (Park et al. 1999. Changes in Body Composition in Mice DuringFeeding and Withdrawal of Conjugated Linoleic Acid. Lipids 34, 243-248).

Unconjugated linoleic acid has been found to have adverse effects, suchas a stimulating effect on breast cancer. The antimicrobial effect ofunconjugated linoleic acid is also generally known.

CLA can be prepared chemically or enzymatically by isomerizing linoleicacid. Natural CLA is formed from i.a. multi-unsaturated fatty acids as aresult of the action of the bacterium Butyrivibrio fibrisolvens in therumen of ruminants, from which it is secreted into milk and meat, whichhave been found to be the best sources of CLA.

It has been noted the amount of CLA obtained from food has decreasedconsiderably during the past decade. It has been calculated in foodcontent analyses that in the 1970's, an average diet contained about0.45 g of CLA/day. As the use of milk and dairy products has declined,the average intake is nowadays 0.25 g of CLA/day. Increase of the amountof natural CLA in food is very important in respect of public healthsince duplication of the CLA intake would, according to research,decrease the risk of cancer, for instance.

As regards food products, the importance of milk as the source of CLAhas been highlighted in several studies. For example, according to aFinnish demographic study (Knekt et al., oral communication), the use ofmilk reduced the risk of breast cancer. Nowadays the CLA content of milkfat varies periodically to a considerable extent (2.4 - 28.1 mg/g offat) depending on the feed quality.

It has been found that intestinal beneficial microbes form CLA. Inparticular, the rumen bacterium Butyrivibrio fibrisolvens and itsisomerase enzyme have been studied. However, this bacterium is anaerobicto such an extent that CLA production by means of it is not feasible inindustrial scale since it is difficult and uneconomical to arrange thestrict anaerobic conditions required by the strain (U.S. Pat. No.5,856,149, Pariza et al.).

It has been found that the species Propionibacterium agnes forms CLA,too, but this pathogenic strain also produces reductase enzyme, whichreduces the produced CLA to other fatty acids (Verhulst et al., System.Appl. Microbiol. 9 (1987) 12-15).

It is further generally known that certain propionic acid bacteria canconvert linoleic acid into its conjugated cis-9,trans-11 form. Inaddition, it is general knowledge that the conversion of free linoleicacid into CLA is more effective than that of triglyceride fatty acid.However, free linoleic acid has a growth inhibiting effect on propionicacid bacteria already in relatively small concentrations, which has sofar prevented large scale production of conjugated linoleic acid andparticularly the cis-9,t-11 form thereof.

U.S. Pat. No. 5,856,149, Pariza and Yang, describes a process forproducing cis-9,trans-11 fatty acid by conversion of unconjugatedunsaturated (double bonds at positions 9 and 12) fatty acid by means ofthe Lactobacillus reuterii strain, preferably the L. reuterii PYR8strain. The publication describes isolation of CLA-producing strains andstates that only 4 out of 45 isolated strains had the desired linoleateisomerase activity, i.e. these were able to produce CLA from linoleicacid. The publication does not mention the inhibiting effect of freelinoleic acid on bacterial growth nor does it set forth a solution foravoiding this problem.

In Production of conjugated linoleic acid by daily starter cultures, J.Appl. Microbiol. 85 (1998), PP. 95-102, J. Jiang, L. Björck and R.Fonden describe the ability of propionic acid bacteria to convertlinoleic acid into CLA. Having noticed that mature cheeses containhigher amounts of CLA than other dairy products, Jiang et al. studiedthe ability of 19 different starter bacteria to convert linoleic acidadded to a culture medium into CLA. They studied the ability of 7lactobacillus strains, 4 lactococcus strains, 2 streptococcus strainsand 6 propionic acid bacteria to produce CLA from linoleic acid on MRS,milk and Na lactate culture media. In addition, different linoleic acidconcentrations were studied by adding linoleic acid to MRS broth inaqueous solution of Tween 80 detergent. Only a few propionic acidbacteria out of the analyzed bacteria showed bioconversion activity;three out of six strains showed activity, i.e. Propionibacteriumfreudenreichii ssp. freudenreichii PFF and PFF6 and P. freudenreichiissp. shermanii PFS. The maximal production of 265 μg/ml of CLA from anoriginal linoleic acid concentration of 750 μg/ml was achieved with thePFF6 strain. The produced CLA contained 70 to 90% of biologically activec9,t11 isomer. None of the lactobacilli, lactococci or streptococci wasfound to produce CLA.

The best propionic acid bacterium, the PFF6 strain, was thus able toconvert only 35% of the added linoleic acid into CLA by the techniquedescribed by Jiang et al. The researchers found that the CLA productionof the propionic acid bacteria correlated positively with theirtolerance with respect to free linoleic acid. Consequently, this studyconfirmed the generally known fact that linoleic acid has anantimicrobial effect that inhibits bacterial growth. The publicationstates that the effect of antimicrobial fatty acids can be reduced byusing surface-active agents, such as detergents, e.g. Tween 80, orproteins. However, such studies have not been carried out and thepublication does not disclose feasible, useful techniques.

WO 99/29886, J. Jiang, L. Björck and R. Fonden, is partly based on theresearch results described in the above-mentioned article. Theapplication relates to the use of certain bacteria useful in foodproduct applications in in vitro production of CLA. In addition toPropionibacterium freudenreichii ssp. freudenreichii and P.freudenreichii ssp. shermanii, Bifidobacterium breve is mentioned as asuitable bacterium. According to the publication, fermentation can becarried out in the presence of an emulsifying agent, such as Tween 80and lecithin. The examples of this publication do not describe the useof an emulsifying agent, either, and the result given as the best resultis the same as in the above-mentioned article: 246.4 μg/ml ofbiologically active c9,t11 isomer was obtained from an original linoleicacid concentration of 750 μg/ml using the PFF6 strain. Thus the yieldwas below 33%.

Finnish patent 88856 describes a process for preparing a fermented foodproduct which contains living microorganisms and is mainly based on oatbran. Oat bran is fermented either as such or after heat treatment, andlactic acid bacteria, in particular Lactobacillus acidophilus, are usedas microorganisms. The object of the invention described in thepublication is to utilize the high fibre content of oat in new kind offood product. As an example the publication gives a yoghurt-type snack.The publication does not mention linoleic acid or conjugated linoleicacid produced therefrom.

Consequently, there is still a clear need for new processes forproducing conjugated linoleic acid. When CLA is to be produced by meansof microbiological processes, it is essential how the problems relatedto the toxicity and antimicrobial effect of external linoleic acid canbe minimized or avoided. Processes where new raw materials can beutilized in the CLA production are also very welcome.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is based on utilization of grain as the source oflinoleic acid. As regards different grain species, oat is deemed to bethe preferred source of linoleic acid.

The invention thus relates to a process for preparing conjugatedlinoleic acid (CLA) from linoleic acid, the process utilizing grainincluding linoleic acid as the source of linoleic acid.

The process of the invention preferably comprises two steps where grainfat is hydrolyzed to release linoleic acid therefrom and the releasedlinoleic acid is isomerized into conjugated linoleic acid by means ofmicrobes.

The invention also relates to the use of grain in the preparation ofconjugated linoleic acid.

The invention further relates to products prepared by the process of theinvention and to their use as such or in the preparation of functionalsubstances.

DETAILED DESCRIPTION OF THE INVENTION

The process of the invention for preparing conjugated linoleic acid bymeans of a microorganism is characterized in that grain fat includinglinoleic acid is hydrolyzed and the linoleic acid released by hydrolysisis isomerized into conjugated linoleic acid by means of a microorganism.

The invention is thus based on the use of grain as the source oflinoleic acid. According to the invention, linoleic acid is releasedfrom grain by means of a hydrolysis reaction of fat. In connection withthe present invention it has been surprisingly found that when grainmaterial is used as the source of linoleic acid as described in thisapplication, linoleic acid does not inhibit the isomerization reaction.When grain, particularly oat, is used as the starting material, it canbe ensured that linoleic acid is available to microbes for the wholeduration of isomerization without the linoleic acid preventing thefunctioning of the bacteria.

The grain used as the source material can be any grain that includeslinoleic acid and is suitable for use as the starting material of anedible product. Oat, barley, rye, wheat and malts prepared therefrom canbe mentioned as examples. Suitable raw materials include untreated andtreated grains and fractions prepared therefrom.

According to the present invention, oat is the most preferred startingmaterial. The linoleic acid content of oat is about 2 to 4% of drysolids, and most of the linoleic acid is bound to diglycerides andtriglycerides. Oat also contains lipase enzyme which degradesdiglycerides and triglycerides into free fatty acids. Considering theobjects of the invention, oat is an advantageous raw material due to itshigh linoleic acid content and natural lipase activity.

The natural lipase activity of grain, particularly oat, can be utilizedin the hydrolysis reaction of fat. The enzyme activity required by thereaction can also be added externally. The CLA yield can be improvedboth in the case of oat and especially other grain by adding lipaseenzyme to the reaction according to the need.

The enzymatic hydrolysis of oat fat or fat of another grain can also befacilitated by pretreatment. One advantageous pretreatment method ismalting, which can be used to produce lipase activity in grain. Othersuitable pretreatments include crushing, grinding, pulverizing, anddissolution in a suitable solvent, particularly in water or anotherliquid medium.

The lipolysis of oat, for example, can be initiated by crushing oatgrains and adding water to the crushed oat. Free linoleic acid formed inthe lipolysis partly binds to other components of oat, which decreasesthe amount of linoleic acid available to the isomerization reaction, andwhich should thus be avoided.

The problem can be reduced or even eliminated by the selection ofsuitable reaction conditions. In the selection of conditions, it is mostessential to prevent the characteristic pH decrease of the oat materialby keeping the pH at a sufficiently high level during the linoleic acidisomerization step. A suitable pH is 6.5 to 9, for instance. Preferably,the pH is adjusted to a level of about 7.0 to about 9.0, preferably to alevel of about 8.0 to about 8.5. This pH regulation prevents linoleicacid from binding to the oat material, which appears as an advantageouseffect on the isomerization reaction. It is important that the pHdecrease in the isomerization mixture is caused by the oat materialitself and not by fermentation. Thus the isomerization reaction does notrequire conventional fermentation, i.e. acidification, but it involvesbioconversion. Most of the CLA formed in the isomerization reaction iscis-9,t-11 isomer.

The linoleic acid released according to the invention (from oat) is usedin the CLA production. The isomerization reaction can be carried outchemically, enzymatically or microbiologically, for example. Theconversion of linoleic acid into CLA is preferably carried outmicrobiologically. In bioconversion, any bacterium that has the abilityof converting linoleic acid into CLA may be used, such as the bacteriamentioned above in the description of the background art. However,isomerization is preferably carried out by means of beneficial bacteriasuitable for use in foodstuffs applications, in particular by means ofpropionic acid bacteria. Strains belonging to the speciesPropionibacterium freudenreichii, and in particular strains belonging tothe subspecies P. freudenreichii ssp. freudenreichii and P.freudenreichii ssp. shermanii are suitable, for example.

Isomerization is carried out in a manner known per se. The componentsand conditions of the isomerization mixture are selected according tothe requirements of the strain to be used so as to obtain an optimal CLAyield. After the publication of the present invention, selection ofsuitable reaction parameters will be part of the know-how of a personskilled in the art.

The fat hydrolysis and isomerization steps can be carried out inparallel, i.e. simultaneously, or consecutively in different vessels orin the same vessel. Simultaneous performance of the steps in one vesselis considered to be an advantageous alternative due to the ease of theprocess.

In a particularly preferred embodiment, beneficial bacteria, preferablypropionic acid bacteria are added to the ground oat, in which case thelinoleic acid released in the lipolysis is directly reacted with thebeneficial bacteria, which isomerize the linoleic acid into conjugatedlinoleic acid. By adjusting the process conditions to suit the lipolysisand isomerization reaction, formation of CLA in considerable amounts inthe mixture can be obtained. Water or another suitable medium,particularly a liquid medium, can be used to facilitate mixing. Inconnection with the present invention, water, for example, has been usedas the medium so that the dry solids content of the oat mixture is 5%,in which case a CLA formation of about 1% of the oat dry solids and ofabout 10% of the oat fat has been obtained.

By combining grain properties with the use of a bacterium capable ofisomerization as a catalyst in the isomerization reaction, two of thegreatest problems related to the CLA production can be avoided: toxicityof linoleic acid and its poor solubility in water. The ability of a CLAproducing strain is preferably combined with a material which containslinoleic acid and lipase and which in the ground form provides linoleicacid for the CLA production without any other additions. Such a materialamong grain is oat. When materials with no lipase activity, such aswheat, are used, external lipase activity can be added or formed throughmalting. According to the present invention, the use of detergentsneeded to “dissolve” external linoleic acid or other harmful additivescan be avoided.

The CLA containing (oat) bacteria mixture prepared according to theinvention can be used as such, it can be added and used in thepreparation of food products and similar functional products, andvarious CLA containing fractions can be isolated therefrom. The CLAformation can also occur simultaneously with the preparation of a foodproduct. When different products are formed, functional properties ofCLA, beneficial bacteria and/or grain, such as oat, can be utilized inthe products in a desired manner.

Embodiments where conjugated linoleic acid is isolated from theisomerization mixture are considered preferred embodiments. When thefunctional effects of both conjugated linoleic acid and bacterial cellsare to be utilized, they can be recovered together, concentrated andpossibly dried or lyophilized. When water is used as the medium, CLA canbe bound to the (oat) bacterial solids by decreasing the pH. Accordingto the invention, conjugated. Linoleic acid can be bound to the solidsby adjusting the pH of the reaction mixture to about 3 to 9, preferablyto a value below 7.0, most preferably to about 4 to 6.

In the present document, the term food is used in a broad sense coveringall edible products which can be in solid, gelled or liquid form, andcovering both ready-to-eat products and products to which the product ofthe invention is added in connection with consumption, as an additive orto be a constituent component of the product. For instance, foods can beproducts of dairy industry, meat processing industry, food processingindustry, beverage industry, bakery industry, confectionery industry andfeed industry. Typical products include milk and milk products, such asyoghurt, curdled milk, curd cheese, sour milk, buttermilk and otherfermented milk beverages, unripened cheeses and ripened cheeses, snackfillings, etc., beverages, such as whey beverages, fruit beverages,beers and soups. Products of the feed industry constitute anotherimportant group.

Preferred applications include lyophilized products, such as CLA and oatcontaining propionic acid bacterium capsules and powders, and productswhose CLA content has been increased utilizing the activity of thepropionic acid bacterium. Products including both CLA and oatcomponents, e.g. β-glucan, are particularly preferable, i.e. productsexpressing the functional effects of both ingredients. An importantadditional advantage of the present invention is that conjugatedlinoleic acid can be formed in oat products, and thus the nutritionalvalue of oat can be increased.

The invention will be described in greater detail by means of thefollowing examples. These examples are only intended to illustrate theinvention, not to restrict its scope in any way.

REFERENCE EXAMPLE 1

CLA Concentration of a Product Based on Fermented Oat Bran

The fatty acid content and the concentrations of oleic acid, linoleicacid and CLA of the fermented products described in Finnish patent 88856were determined as follows. Samples were taken from commercial products,Yosa wild berry and Yosa plum, produced by Bioferme Oy, Finland, and fatwas isolated from them by direct saponification and diethyl ether hexaneextraction. Methyl esters of fatty acids were prepared by a processcatalyzed by sulphuric acid. Table A shows the fatty acid content of thesamples in per cents (%) of the total amount of fatty acids, and Table Bshows the oleic acid, linoleic acid and CLA (c9,t-11) concentrations asmg/g of sample. Yosa plum and Yosa wild berry products contained hardlyany CLA. The very small CLA residues may have resulted from theinfluence of the analysis methods or they may be isomers of linoleicacid (C_(18:3)) which elute in the gas chromatographic analysis nearCLA. TABLE A Fatty acid content of Yosa samples, in percent (%) of thetotal amount of fatty acids Palmitic Stearic Oleic Linoleic LinolenicCLA acid acid acid acid acid (c-9, t-11) Yosa C_(16:0) C_(18:0)C_(cis-9-18:1) C_(18:2) C_(18:3) C_(18:2) Plum 16.07 1.21 32.05 39.632.04 0.05 Wild 15.58 1.14 30.93 39.45 3.68 0.07 berry

TABLE B Oleic acid, linoleic acid and CLA concentrations of Yosasamples, mg/g of sample Oleic acid Linoleic acid CLA (c-9, t-11) Yosamg/g of sample mg/g of sample mg/g of sample Plum 1.77 2.15 0.003 Wildberry 1.65 2.08 0.004

EXAMPLE 1

CLA Production in an Oat/Water Mixture by Means of Propionic AcidBacteria.

To prove the effect of the process according to the invention, a testwas performed where propionic acid bacteria cells were added to anoat/water mixture for use as an isomerization catalyst. Oatmeal producedby grinding untempered oat (variety Lisbeth) through a screen of 0.5 mmwas used in the test. A 5% water mixture (w/v) was prepared from theoatmeal and the mixture was homogenized by an Ultra Turrax apparatus forabout two minutes.

Two propionic acid bacteria strains were used in the test, i.e.Propionibacterium freudenreichii subsp. shermanii JS (PJS) andPropionibacterium freudenreichii subsp. freudenreichii 131 (P131).

Culturing of PJS cells for the test was carried out as described inRainio, A., Vahvaselkä, M., Suomalainen, T. ja Laakso, S., Reduction oflinoleic acid inhibition in production of conjugated linoleic acid byPropionibacterium freudenreichii ssp. shermanii, Can. J. Microbiol. 47(2001), 735-740. The P131 strain was cultured in MRS liquor (LabM). Thecells were centrifuged (6000 rpm, 20 min) to separate them andelutriated in a small amount of peptone salt solution, which contained0.1% of bacteriological peptone (LabM), and 0.85% of NaCl. The cellsuspension was added to the oatlwater mixture (a′ 100 ml) to obtain acell concentration of about 1×10¹⁰ cfu/ml. The pH of the mixture wasadjusted to 7.0 by 1 M NaOH solution, and the hydrolysis andisomerization reaction was allowed to occur at 25° C. During the first17 hours, the oat/water mixture was allowed to hydrolyze without pHregulation, as a result of which the pH decreased to approximately 4.8.After this, the pH of the mixture was raised to 8.0 by NaOH solution andthe adjustment was repeated approximately every 1.5 to 2 hours for abouteight hours. The pH of the mixture thus remained approximately between7.5 and 8.0. After this, isomerization was continued without pHregulation until the total time was about 40 hours.

By comparison, a test was carried out where a corresponding volume ofpeptone salt solution was added to the oat/water mixture instead of thePJS or P131 cell suspension. During the first 17 hours, the pH of themixture decreased to about 5.4. After this, the pH was adjusted as inthe test described above.

In the test, release of linoleic acid as a result of the activity of thenatural lipase enzyme of oat and isomerization of this free linoleicacid into CLA by means of microbe cells were followed. Samples of 0.5 mlwere taken from the oat/water mixture, and these were cold-dried beforethe fatty acid analysis. The amounts of different fatty acids weredetermined from the samples by means of gas chromatography. The analysisof fatty acids was carried out as described in Suutari M., Liukkonen, K.and Laakso, S., Temperature adaptation in yeasts: the role of fattyacids, J. Gen. Microbiol. 136 (1990), 1469-1474. The fatty acidsincluded in the samples were identified by comparing their retentiontimes to the retention times of known fatty acid standards. Conjugatedlinoleic acid was identified utilizing a preparation from Sigma, whichwas a mixture of cis and trans-9,11 and cis and trans-10,12 isomers ofCLA. A methyl ester of C17:0 fatty acid (heptadecanoic acid methylester, Sigma) was used as internal standard in the quantification offatty acids.

Samples of 1.5 ml, which were cold-dried, were taken from the oat/watermixture for lipid class analysis of the fatty acids. The lipid classanalysis was carried out as described in Liukkonen, K. H., Montfoort, A.and Laakso, S., Water-induced lipid changes in oat processing, J. Agric.Food Chem. 40 (1992) 126-130.

The amounts of linoleic acid and CLA formed were calculated as afunction of reaction time per solids sample. The test was carried out inthe oat/water mixture both in the presence of propionic acid bacteria(PAB) and without them. The results are presented in Table 1. TABLE 1Amounts of linoleic acid and CLA formed as a function of reaction timecalculated per solids sample. Linoleic acid (mg/g CLA (mg/g of dry ofdry solids) solids) Time PJS P131 no PHB PJS P131 no PHB (h) cells cellscells cells cells cells  0 41.9 36.3 43.4 <0.1 <0.1 <0.1 17 40.7 — — 0.6— — 21, 5 36.3 — — 4.8 — — 25 33.6 — — 6.5 — — 41 32.3 32.7 42.3 7.6  0.7 <0.1

The CLA formation thus required the functioning of propionic acidbacterium as isomerization catalyst in the oat/water mixture. When thePJS strain was used, considerable amounts of CLA were formed, 7.6 mg/gof dry solids. This amount was 7.3% of the fat included in oat.

Table 2 shows the distribution of linoleic acid and CLA into differentlipid classes when the oat/water mixture was incubated together with PJScells. PL=polar lipids, TG=triglycerides, DG=diglycerides and FFA=freefatty acids. TABLE 2 Distribution of linoleic acid and CLA intodifferent lipid classes (%) during the test. Time Compound PL TG DG FFA 0 h linoleic acid 12 81 3 4 17 h linoleic acid 9 49 5 37 41 h linoleicacid 12 40 5 43 41 h CLA 4 8 1 87

The results show that at the beginning of the test, most of the linoleicacid was bound to triglycerides and only 4% of it was in the form offree fatty acid. However, after a 17-hour hydrolysis, nearly 40% of thelinoleic acid had been released from the triglycerides. The forming CLAwas mostly (nearly 90%) in the form of free fatty acid. At least 80% ofthe CLA formed was cis-9,trans-11 isomer.

The concentrations of living propionic acid bacterial cells weredetermined using buffered sodium lactate agar, which contained 0.5% oftryptone (LabM), 1% of yeast extract (LabM), 16.8 mUl of 50% Na lactate(Merck), 1% of disodium salt of P-glycerophosphate (Merck) and 1.5% ofagar (LabM). The plates were incubated anaerobically at 30° C. for 6days. At the beginning of the test, the PJS concentration was 9.0×10⁹cfu/ml and after 20 hours 7.5×10⁹ cfu/ml. On the basis of the results,the propionic acid bacterial cells thus did not grow in the oat/watermixture during the reaction.

The pH of the oat/water mixture tended to decrease rapidly regardlesswhether propionic acid bacterium had been added to the mixture or not.The pH decrease is caused by dissolution of acid components of oat inwater. The process does not thus require that the cells be able toferment oat, whereby the organic acids formed would decrease the pH.

EXAMPLE 2

CLA Production from Other Grain Species by Propionic Acid Bacteria.

Example 1 was repeated using barley and rye instead of oat.Propionibacterium freudenreichii subsp. shermanii JS (PJS) cells wereused as the propionic acid bacterium. On the basis of the results, CLAproduction in a mixture of barley or rye in water was considerablyweaker than in the oat/water mixture; 0.91 mg of CLA / g of dry solidswas formed in barley and 0.83 mg in rye during a 41-hour incubationwhere the pH was adjusted to 8.0 between 17 and 25 hours. The poorerresults are partly explained by the fact that the linoleic acidconcentration of these grain materials is smaller than that of oat.Furthermore, it is known that they do not include lipase activitywithout germination. However, based on the results, it is clear that theprocess according to the invention also functions in other grainmaterials. Formation of free linoleic acid can be enhanced by addingexternal lipase activity to the reaction mixture, in which case theprocess yield can be improved significantly from the values given above.

EXAMPLE 3

Effect of pH on CLA Formation.

The effect of pH of the oat/water mixture on the CLA formation wasanalyzed by the following tests:

-   -   the pH was not adjusted at all    -   the pH was adjusted to 8.0 between 0 to 8 hours (thus the test        did not include a separate fat hydrolysis step)    -   the pH was adjusted to 7.0 between 17 to 25 hours    -   the pH was adjusted to 8.0 between 17 and 25 hours    -   the pH was adjusted to 8.5 between 17 to 25 hours    -   the pH was adjusted to 9.0 between 17 and 25 hours.

In all the above-mentioned tests, the PJS bacteria strain was added tothe oat/water mixture as described in example 1. The other testarrangements were also the same.

In addition, a test was performed in a fermenter, where the pH of theoat/water mixture was kept at 8.5 during the whole isomerization step(between 17 and 41 h) by means of automatic addition of NaOH solution.In the 17-hour lipolysis step preceding it, the pH decreased to 4.7. Thevolume of the oat/water mixture was 1.5 litres, temperature 25° C. andmixing speed 100 rpm. The concentration of living PJS cells was 1.1×10¹⁰cfu/ml at the beginning of the test and 8.4×10⁹ cfu/ml at the end of thetest.

The results are shown in Table 3. According to the results, the CLAformation was effective when the pH of the oat/water mixture wasadjusted to between 8.0 and 8.5 after the lipase enzyme had releasedlinoleic acid. This proceeded best at a pH lower than that of theisomerization reaction. The fastest and greatest CLA formation wasachieved when the pH of the oat/water mixture was kept at 8.5 bycontinuous regulation during the whole isomerization step. This reflectsthe importance of even pH level suitable for the isomerization reactionto the process. TABLE 3 Influence of the pH of the oat/water mixture onthe CLA formation. CLA (mg/g of dry solids) 25 h 41 h pH not adjusted0.9 pH adusted to a approx. 8.0 between 0-8 h 3.5 PH adjusted to approx.7.0 between 17-25 h 3.9 PH adjusted to approx. 8.0 between 17-25 h 6.57.6 pH adjusted to approx. 8.5 between 17-25 h 7.9 pH adjusted toapprox. 9.0 between 17-25 h 6.0 pH kept at 8.5 by automatic regulation8.2 9.3 between 17-41 h

EXAMPLE 4

Concentration of Produced CLA into Oat Dry Solids by Means of pHDecrease.

CLA production in an oat/water mixture was performed according toexample 1 by means of PJS cells. After this, the pH of the oat/watermixture was adjusted to 8.0 by NaOH solution or to 4.5 by HCl solution.The mixtures were centrifuged and CLA concentrations were determinedfrom supernatants and oat bacteria masses. The CLA distribution was asfollows: at the pH of 8.0, 85% of the CLA was in the solids and 15% inthe liquid step, at the pH of 4.5, 100% of CLA was in the solids. Theresult provides a process by means of which, utilizing the pH decrease,CLA can be made to concentrate into the solids formed by oat andbacterial cells, and thus the CLA is not removed together with themedium.

1. A process for preparing conjugated linoleic acid by microorganisms,characterized by hydrolyzing oat fat and isomerizing the linoleic acidreleased in the hydrolysis into conjugated linoleic acid by themicroorganisms.
 2. A process according to claim 1, characterized in thatthe grain is untreated oat, pretreated oat or an oat fraction.
 3. Aprocess according to claim 1, characterized in that the fat hydrolysisis caused by the enzyme activity of oat.
 4. A process according to claim1, characterized in that the fat hydrolysis is carried out by addingexternal enzyme activity.
 5. A process according to claim 1,characterized in that isomerization is carried out by a beneficialbacterium (bacteria).
 6. A process according to claim 1, characterizedin that the beneficial bacterium is a propionic acid bacterium.
 7. Aprocess according to claim 6, characterized in that the propionic acidbacterium is a strain belonging to the species Propionibacteriumfreudenreichii, preferably a strain belonging to its subspeciesPropionibacterium freudenreichii ssp. freudenreichii orPropionibacterium freudenreichii ssp. shermanii.
 8. A process accordingto claim 7, characterized in that the propionic acid bacterium isPropionibacterium freudenreichii ssp. shermanii JS, DSM
 7067. 9. Aprocess according to claim 1, characterized in that isomerization iscarried out at a pH of about 6.5 to 9.5.
 10. A process according toclaim 9, characterized in that isomerization is preferably carried outat a pH of about 7.0 to 9.0, more preferably at a pH of about 8.0 to8.5.
 11. A process according to claim 1, characterized in that thehydrolysis and isomerization steps are carried out consecutively.
 12. Aprocess according to claim 1, characterized in that the hydrolysis andisomerization steps are carried out in parallel.
 13. A process accordingto claim 1, characterized in that the preparation of conjugated linoleicacid occurs in connection with the preparation of a food product.
 14. Aprocess according to claim 1, characterized in that mainly cis-9,trans-11 isomer of conjugated linoleic acid is formed therein.
 15. Aprocess according to claim 1, characterized in that conjugated linoleicacid is fixed to solids by adjusting the pH of the reaction mixture toabout 3 to 9, preferably to a value lower than 7.0, most preferably toabout 4 to
 6. 16. A process according to claim 1, characterized in thatconjugated linoleic acid is isolated from the reaction broth andpossibly dried.
 17. A process according to claim 1, characterized inthat conjugated linoleic acid, bacterial cells and the oat material usedas starting material, which is preferably oat material are concentratedand possibly dried.
 18. A process according to claim 17, characterizedin that linoleic acid, bacterial cells and oat material used as thestarting material are recovered, concentrated and lyophilized.
 19. Oatfor use in the preparation of conjugated linoleic acid.
 20. A processfor preparing conjugated linoleic acid from linoleic acid, characterizedin that oat is used as the source of linoleic acid.